Bo Bin Lee

Aberrant Methylation of APC, MGMT, RASSF2A, and Wif-1 Genes in Plasma as a Biomarker for Early Detection of Colorectal Cancer

Purpose: To identify epigenetic molecular makers in plasma for the early detection of colorectal cancer. Experimental Design: We retrospectively analyzed the methylation status of 10 genes in fresh-frozen tissues and corresponding plasma samples from 243 patients with stage I and II sporadic colorectal cancer, 276 healthy individuals, and plasma from 64 colorectal adenoma patients using methylation-specific PCR. The methylation score (Mscore) was used to find molecular markers with high sensitivity and specificity. Results: Of the 243 colorectal cancer tissues, methylation was detected in 18% for p14, 34% for p16, 27% for APC, 34% for DAPK, 32% for HLTF, 21% for hMLH1, 39% for MGMT, 24% for RARβ2, 58% for RASSF2A, and 74% for Wif-1. Receiver operator characteristic curve analysis in plasma from 243 patients with cancer and 276 healthy individuals showed that the M score of any single gene had a sensitivity of <40% after controlling for age, sex, and tumor location. The specificity of the M score was not different between multigene and single gene analyses, but the sensitivity of the M score was significantly increased by multigene analysis. For all patients, the M score in a model including APC, MGMT, RASSF2A, and Wif-1 genes had a sensitivity of 86.5% and a specificity of 92.1% when 1.6 was used as a cutoff. In this model, the M score had a positive predictive value of 90.6% and a negative predictive value of 88.8%. Conclusion: The present study suggests that tumor-specific methylation of APC, MGMT, RASSF2A, and Wif-1 genes might be a valuable biomarker in plasma for the early detection of colorectal cancer. (Clin Cancer Res 2009;15(19):6185–91)

Association of the SUV39H1 histone methyltransferase with the DNA methyltransferase 1 at mRNA expression level in primary colorectal cancer.

This study was aimed at investigating the involvement of the SUV39H1 histone methyltransferase on the epigenetic change of euchromatic promoter in colorectal cancer. We retrospectively analyzed the mRNA levels of SUV39H1 and the promoter methylation of the p14ARF, p16INK4a and HLTF genes as well as the mRNA levels of DNA methyltransferase 1 (DNMT1) in fresh frozen tissues from 219 colorectal cancer patients. The mRNA levels of the SUV39H1 and DNMT1 were assessed via quantitative real‐time PCR and the methylation profiles of the CpG islands were determined using methylation‐specific PCR. The mRNA levels of SUV39H1 and DNMT1 were elevated in 25% and 42% of 219 colorectal cancers, respectively. The hypermethylation of the p14ARF, p16INK4a and HLTF genes occurred in 36%, 51% and 34% of the patients. The elevated mRNA levels of SUV39H1 were not associated with the hypermethylation of the 3 genes. However, the mRNA levels of DNMT1 were significantly different between patients with elevated mRNA levels of SUV39H1 and those without (1.62 ± 0.84, 0.91 ± 0.81, respectively; p = 0.007). Patients with elevated mRNA levels of SUV39H1 showed a higher prevalence of DNMT1 elevation than those without (61 vs. 35%, p = 0.0008). Patients with an elevated mRNA level of SUV39H1 had a 2.71 (95% CI = 1.09–4.48, p = 0.002) times greater risk of an elevated mRNA level of DNMT1, after controlling for age and gender. In conclusion, the present study suggests that SUV39H1 is significantly associated with DNMT1, but not with euchromatic promoter methylation in colorectal cancer.

CpG island hypermethylation of E-cadherin (CDH1) and integrin α4 is associated with recurrence of early stage esophageal squamous cell carcinoma

The prognosis of esophageal squamous cell carcinoma (ESCC) patients remains very poor, which is partially due to a high rate of recurrence. This study was aimed at identifying a recurrence‐associated epigenetic prognostic marker in patients with ESCC. We retrospectively analyzed the CpG island hypermethylation of the p16, Wif‐1, sFRP1, integrin α4, CDH1, DAP kinase and RARβ2 genes in 251 ESCCs. The methylation status was determined by methylation‐specific PCR. Hypermethylation was detected in 52% for p16, 25% for RARβ2, 43% for CDH1, 21% for integrin α4, 57% for sFRP1, 38% for DAP kinase and 35% for Wif‐1. Recurrence was observed in 131 (52%) of the 251 cases. For stage I cancers, CDH1 methylation was associated with a high risk of recurrence (OR = 5.26, 95% CI = 1.48–18.67; p = 0.01) and a poor recurrence‐free survival after surgery (HR = 3.13, 95% CI = 1.21–8.09; p = 0.02). The hazard of failure after recurrence was about 13.17 (95% CI = 2.46–70.41; p = 0.003) times higher in patients with Wif‐1 methylation than in those without. For stage II cancers, integrin α4 methylation was associated with an increased risk of recurrence (OR = 3.03, 95% CI = 1.09–8.37; p = 0.03) and a poor recurrence‐free survival (HR = 2.12, 95% CI = 1.13–3.98; p = 0.03). In conclusion, the present study suggests that hypermethylation of CDH1 and integrin α4 genes may be used as recurrence‐associated prognostic indicators in stage I and stage II ESCCs, respectively.

Association of interindividual differences in p14ARF promoter methylation with single nucleotide polymorphism in primary colorectal cancer

CpG island hypermethylation has been reported at the promoter region of many tumor suppressor genes in colorectal cancers. However, there are significant interindividual differences in the degree of DNA methylation in colorectal cancers. The objective of the current study was to understand whether single nucleotide polymorphisms (SNPs) around the promoter of a gene are implicated in the interindividual differences of CpG island hypermethylation.

Association of RASSF1A and p63 with Poor Recurrence-Free Survival in Node-Negative Stage I–II Non–Small Cell Lung Cancer

Purpose: This study was aimed at analyzing the recurrence-related prognostic significance of 12 candidate molecular biomarkers in node-negative stage I–II non–small cell lung cancer (NSCLC). Experimental Design: We retrospectively analyzed promoter methylation of eight genes using methylation-specific PCR in formalin-fixed and paraffin-embedded tissues from 328 node-negative stage I–II NSCLCs. The expression of Bcl-2, E-cadherin, p53, and p63 proteins was also assessed by immunohistochemistry. Results: Recurrence was found in 145 (44%) of 328 node-negative stage I–II NSCLCs with a median follow-up period of 6.2 years. No association was found between recurrence and alteration of individual biomarker in univariate analysis. We defined recurrently divergent groups on the basis of recursive partitioning analyses for 12 biomarkers and found a significant association of co-alteration of RASSF1A and p63 with poor recurrence-free survival (RFS). Cox proportional hazards analysis showed that hypermethylation of RASSF1A and negative expression of p63 was associated with poor RFS [HR, 1.93; 95% confidence interval (CI), 1.13–5.47; P = 0.009] compared with those without co-alteration of RASSF1A and p63, after adjusting for age, adjuvant therapy, histology, and tumor size. Random forest classifier including RASSF1A and p63 showed best performance in the prediction of recurrence in node-negative stage I–II NSCLCs: area under receiver operator characteristic curve for random forest was 0.91 and error rate for the model was 17%. Conclusion: The present study suggests that RASSF1A and p63 may be independent prognostic indicators for RFS in node-negative stage I–II NSCLCs. Clin Cancer Res; 19(5); 1204–12. ©2012 AACR.

HOXA9 inhibits migration of lung cancer cells and its hypermethylation is associated with recurrence in non‐small cell lung cancer

This study was aimed at understanding the clinicopathological significance of HOXA9 hypermethylation in non‐small cell lung cancer (NSCLC). HOXA9 hypermethylation was characterized in six lung cancer cell lines, and its clinicopathological significance was analyzed using methylation‐specific PCR in 271 formalin‐fixed paraffin‐embedded tissues and 27 fresh‐frozen tumor and matched normal tissues from 298 NSCLC patients, and Ki‐67 expression was analyzed using immunohistochemistry. The promoter region of HOXA9 was highly methylated in six lung cancer cell lines, but not in normal bronchial epithelial cells. The loss of expression was restored by treatment of the cells with a demethylating agent, 5‐aza‐2′‐deoxycytidine (5‐Aza‐dC). Transient transfection of HOXA9 into H23 lung cancer cells resulted in the inhibition of cell migration but not proliferation. Conversely, sequence‐specific siRNA‐mediated knockdown of HOXA9 enhanced cell migration. The mRNA levels of HOXA9 in 27 fresh‐frozen tumor tissues were significantly lower than in matched normal tissues (P < 0.0001; Wilcoxon signed‐rank test). HOXA9 hypermethylation was found in 191 (70%) of 271 primary NSCLCs. HOXA9 hypermethylation was not associated with tumor size (P = 0.12) and Ki‐67 proliferation index (P = 0.15). However, patients with HOXA9 hypermethylation had poor recurrence‐free survival (hazard ratio = 3.98, 95% confidence interval = 1.07–17.09, P = 0.01) in never‐smokers, after adjusting for age, sex, tumor size, adjuvant therapy, pathologic stage, and histology. In conclusion, the present study suggests that HOXA9 inhibits migration of lung cancer cells and its hypermethylation is an independent prognostic factor for recurrence‐free survival in never‐smokers with NSCLC.

Cohypermethylation of p14 in combination with CADM1 or DCC as a recurrence-related prognostic indicator in stage I esophageal squamous cell carcinoma.

The objective of this study was to discover molecular biomarkers associated with the recurrence of esophageal squamous cell carcinoma (ESCC).

Relationship of phospho-pRb (Ser-807/811) level to exposure to tobacco smoke in primary non-small cell lung cancer.

This study was aimed at understanding the effect of smoking on pRb phosphorylation and the clinicopathological significance of phospho-pRb in non-small cell lung cancers (NSCLCs). Phospho-pRb (Ser-807/811) expression was not detected in 149 (39%) of 382 patients, and the mean phospho-pRb (Ser-807/811) level was 5.7%. Squamous cell carcinoma had higher phospho-pRb (Ser-807/811) levels than adenocarcinoma (7.1%+/-10.4% versus 4.7%+/-7.9%; P=0.003). The association between phospho-pRb (Ser-807/811) levels and exposure to tobacco smoke was different according to the statuses of cyclin D1 expression and p16 methylation, suggesting that their statuses might play a role as an effect modifier in the relationship between phospho-pRb (Ser-807/811) levels and exposure to tobacco smoke. In stratified multivariate analysis, phospho-pRb (Ser-807/811) levels were not associated with exposure to tobacco smoke in 38 patients with p16 hypermethylation and cyclin D1 expression >5%, after adjusting for confounding factors. However, in the remaining 344 patients, the mean phospho-pRb (Ser-807/811) levels in patients who had smoked >40 pack years increased by 4.65% (P<0.0001) on average than those who had never smoked. No association was found between the phospho-pRb (Ser-807/811) levels and overall survival. In conclusion, the present study suggests that exposure to tobacco smoke is associated with phosphorylation of pRb in NSCLC patients and its relationship depends on the p16 methylation status and cyclin D1 expression levels.

Overexpression of β-Catenin and Cyclin D1 is Associated with Poor Overall Survival in Patients with Stage IA–IIA Squamous Cell Lung Cancer Irrespective of Adjuvant Chemotherapy

Introduction: This study was aimed at understanding the effect of &bgr;‐catenin and cyclin D1 on overall survival in patients with early‐stage NSCLC and at evaluating if the prognostic effect can be modified by adjuvant chemotherapy. Methods: We retrospectively analyzed the expression of &bgr;‐catenin and cyclin D1 using immunohistochemistry in formalin‐fixed paraffin‐embedded tissues from 576 patients with early‐stage NSCLC. Results: The median duration of follow‐up was 5.1 years. Overexpression of &bgr;‐catenin and cyclin D1 was found in 56% and 50% of 576 cases, respectively. Overexpression of &bgr;‐catenin and cyclin D1 was significantly associated with poor overall survival (p = 0.003 and p = 0.0009, respectively; log rank test) in squamous cell carcinomas, not in adenocarcinomas. The prognostic significance of each protein in the squamous cell carcinomas was limited to stages IA, IB, and IIA. In addition, simultaneous overexpression of &bgr;‐catenin and cyclin D1 in the squamous cell carcinomas synergistically increased hazard ratios (HRs) 15.79 (95% confidence interval [CI] = 1.09–51.23; p =0.04) for stage IA, 10.30 (95% CI = 2.29–46.41; p = 0.002) for stage 1B, and 3.55 (95% CI = 1.22–10.36; p = 0.02) times for stage 2A compared to those without overexpression of the two proteins, after adjusting for confounding factors. In addition, the effect was not dependent on adjuvant chemotherapy. Conclusions: The present study suggests that simultaneous overexpression of &bgr;‐catenin and cyclin D1 may be associated with poor overall survival irrespective of platinum‐based adjuvant chemotherapy in stage IA–IIA squamous cell carcinoma of the lung.

Metformin induces cell cycle arrest at the G1 phase through E2F8 suppression in lung cancer cells

A target molecule responsible for cell cycle arrest by metformin was discovered using a gene chip array in lung cancer cells and the effect of metformin on E2F8 was assessed. The siRNA-mediated knockdown of E2F8 significantly suppressed G1-S progression while ectopic expression of E2F8 relieved metformin-induced G1 arrest. The mRNA levels of p21 were found to be inversely related to those of E2F8 in lung cancer cells while siRNA-mediated knockdown of p21 partly rescued siE2F8-induced arrest of the cell cycle. Metformin had no effect on degradation of E2F8 mRNA. Activation and inhibition of AMPK by AICAR and Dorsomorphin, respectively, did not affect E2F8 suppression by metformin. The clinical significance of E2F8 was analyzed in The Cancer Genome Atlas (TCGA) data. One hundred six (13%) of 848 TCGA lung cancers overexpressed E2F8 mRNA. The overexpression of E2F8 was associated with poor overall survival (adjusted hazard ratio = 1.58, 95% confidence interval = 1.13–2.22; P = 0.008). The present study suggests that metformin may induce cell cycle arrest at the G1 phase by suppressing E2F8 expression in lung cancer cells. In addition, E2F8 may be associated with poor overall survival in lung cancer patients irrespective of histology.